Human Parvovirus B19 DNA Replication Induces a DNA Damage
Response That Is Dispensable for Cell Cycle Arrest at Phase G2/M
Sai Lou,a,bYong Luo,bFang Cheng,bQinfeng Huang,bWeiran Shen,bSteve Kleiboeker,cJohn F. Tisdale,dZhengwen Liu,aand
Department of Infectious Diseases, First Affiliated Hospital, School of Medicine, Xi’an Jiaotong University, Xi’an, Chinaa; Department of Microbiology, Molecular Genetics
and Immunology, University of Kansas Medical Center, Kansas City, Kansas, USAb; ViraCor-IBT Laboratories, Lee’s Summit, Missouri, USAc; and Molecular and Clinical
Hematology Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda,
ruses, in triggering a DDR in ex vivo-expanded primary human erythroid progenitor cells and the role of DNA replication of the
the DDR. Moreover, the DDR per se did not arrest the cell cycle at the G2/M phase in cells with replicating B19V dsDNA ge-
and belongs to the genus Erythrovirus in the family Parvoviridae
(68). B19V infection in healthy adults is self-limiting, but in im-
munocompromised individuals, those with inherited hemolytic
anemia, and pregnant women, B19V infection can cause aplastic
crisis and hydrops fetalis, which can be fatal (74). B19V infection
63). During B19V infection, nine major mRNA transcripts are
generated by alternative processing of a single precursor mRNA
(50) and encode one large nonstructural 1 (NS1) protein, two
small nonstructural proteins (11-kDa and 7.5-kDa proteins), and
two capsid proteins (VP1 and VP2) (37, 64, 78). The NS1 protein
is essential for B19V DNA replication (78) and is a transactivator
for viral gene expression (22, 25, 55), as well as for the expression
cell cycle arrest (45, 70) and apoptosis (42) of infected EPCs. The
11-kDa protein plays a role in the viral DNA replication (78) and
the 7.5-kDa protein remains unknown. Apart from serving as a
minor structural protein (30), VP1 also contains a unique region
nucleus (75). VP2 is the major structural protein involved in vi-
rion formation (30, 31).
Ex vivo-expanded EPCs are highly permissive for B19V infec-
tion (62, 72) and support an efficient productive B19V infection
erythropoietin-dependent cell lines, e.g., the megakaryoblastoid
cell line UT7/Epo-S1 (S1) (47), support B19V infection with lim-
ited efficiency; however, replication of the B19V genome is in-
uman parvovirus B19 (B19V) is a small nonenveloped virus
with a single-stranded DNA (ssDNA) genome of 5.6 kb (18)
creased approximately 100-fold when S1 cells are cultured under
hypoxic conditions (10). After B19V infection, host cells show
remarkable responses to viral infection. B19V induces a DNA
death (13, 42, 46, 73), among which the B19V-induced DDR is
ectasia-mutated (ATM), ataxia telangiectasia-mutated and Rad3-
related (ATR), and the DNA-dependent protein kinase catalytic
determinant for the DDR induced during B19V infection, and
both the ATR and DNA-PKcs are essential for promoting viral
for triggering the DDR and whether the induced DDR is detri-
DDR involves biochemical pathways that arrest cell cycle pro-
tion of ATR or ATM phosphorylates downstream factors to in-
duce cell cycle arrest and facilitate DNA repair (7, 35). As B19V
infection activates the ATR and ATM signaling pathways, they
presumably contribute to G2/M arrest during B19V infection.
Received 23 April 2012 Accepted 17 July 2012
Published ahead of print 25 July 2012
Address correspondence to Jianming Qiu, firstname.lastname@example.org, or Zhengwen Liu,
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
jvi.asm.orgJournal of Virology p. 10748–10758 October 2012 Volume 86 Number 19
arrest in infected cells, although viral DNA replication is signifi-
inducing G2/M arrest during B19V infection in EPCs through the
interaction between its nuclear localization signal (NLS) domain
and the cellular transcription factors E2F4/E2F5 (70). It is argu-
able that DDR signaling and the interaction between NS1 and
E2F4/E2F5 are redundant for arresting B19V-infected cells at the
bation during B19V infection needs to be confirmed.
We recently demonstrated that hypoxic conditions promote
efficient B19V infection in both EPCs and S1 cells (10). B19V
infection of EPCs in the human bone marrow and fetal liver,
where such hypoxic conditions exist (16, 52, 58, 67). To under-
stand the cause of the DDR and to differentiate the role of the
DDR from that of NS1 in inducing G2/M arrest, in this study we
cultured both S1 cells and EPCs under hypoxic conditions; EPCs
were transduced with lentiviruses expressing individual viral pro-
(dsDNA) form of the B19V ssDNA genome.
MATERIALS AND METHODS
granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral
blood stem cells from healthy donors according to a protocol (02-H-
0160) approved by the National Heart, Lung, and Blood Institute institu-
CD34?cells in Wong medium as previously described (9, 72). Briefly,
conditions until day 7. The cells were then transferred to hypoxic condi-
The S1 cells were cultured as described previously (26) and kept under
hypoxic conditions for 48 h before electroporation, B19V infection, or
The B19V plasma sample (no. P158, ?1 ? 1011genome copies [gc]/
ml) was supplied by the ViraCor-IBT Laboratories (Lee’s Summit, MO).
B19V infection was carried out at a multiplicity of infection (MOI) of
1,000 gc/cell by following the methods previously described (10).
Transfection. The electroporation of S1 cells was performed as previ-
ously described (26). The B19V dsDNA genome (M20) and its derivative
mutants were recovered from SalI-digested pIB19-M20 and its derivative
Plasmid construction. (i) Lentiviral vectors. The DNA coding se-
optimized at GenScript USA Inc. (Piscataway, NJ) to enhance protein
expression efficiency (79). The pLenti-CMV-IRES-GFP-WPRE vector
(36) was used for inserting C-terminally Flag-tagged optimized (opt)
NS1-, 11-kDa protein-, 7.5-kDa protein-, VP1-, and VP2-coding se-
quences at BamHI and BsrGI sites, resulting in the following constructs:
pLenti-optNS1, pLenti-opt11-kDa, pLenti-opt7.5-kDa, pLenti-optVP1,
The B19V P6 promoter-based NS1-expressing lentiviral vector,
pLenti-½ITR-P6-NS1, was constructed as follows: a DNA fragment
from nucleotides 187 to 2628 of the B19V genome, which spans the left
half of the inverted terminal repeat (ITR), namely, ½ITR, the P6 pro-
moter, and the NS1-encoding sequence, with a Flag epitope at the C
terminus, was inserted into ClaI/SalI-digested pLenti-CMV-GFP-
WPRE, which was made by removing the puromycin gene expression
cassette from pLenti-GFP-Puro (Addgene Inc., Cambridge, MA). The
amino acid sequence of B19V NS1 (GenBank accession no.
AAQ91878.1) was aligned with that of adeno-associated virus 2
(AAV2) Rep78 (GenBank accession no. AAC03775.1), which locates
the conserved endonuclease domain (69) and helicase Walker A box site
(43, 69) in the B19V NS1 (see Fig. 2C). Putative transactivation domains
and helicase-A domains and the predicted TAD2 and TAD3 were intro-
ing pLenti-½ITR-P6-NS1(endo-), pLenti-½ITR-P6-NS1(heli-), pLenti-
pLenti-½ITR-P6-RFP was made by replacing the NS1-encoding sequence
(ii) Mutants of pIB19-M20. The B19V infectious clone pIB19-M20
(80), NS1 knockout mutant [pIB19-M20(NS1?)], and VP2 knockout
mutant [pIB19-M20(VP?)] (78) were gifts from Kevin Brown and Ning
Zhi at the NIH. The M20 DNA is a dsDNA form of the B19V ssDNA
B19V genome during virus replication (27). Mutations of the putative
endonuclease and helicase-A domains and the predicted TAD1, TAD2,
and TAD3 of NS1 (see Fig. 2C and 3A) were introduced into pIB19-M20,
creating pIB19-M20(endo?), pIB19-M20(heli?),
M20(mTAD1, mTAD2, and mTAD3).
(iii) pLKO-shRNA vectors. The validated coding sequence for p53
follows: 5=-CCG GTC GGC GCA CAG AGG AAG AGA ATC TCG AGA
TTC TCT TCC TCT GTG CGC CGT TTT TC-3= (clone identifier,
NM_000546.x-1095s1c1). pLKO-shRNA-p53 was made by inserting a
DNA fragment of this sequence into the AgeI/EcoRI-digested pLKO-
GFP-scramble-shRNA vector (Addgene Inc.) as described previously (9).
All the nucleotide numbers of the B19V genome refer to GenBank
accession no. AY386330.
First antibodies used. Rat anti-B19V NS1 (anti-NS1) and anti-11-
kDa protein polyclonal antibodies were previously reported (13, 39).
Mouse anti-7.5-kDa protein polyclonal antibody was made by immuniz-
were obtained commercially: anti-VP1/VP2 antibody (MAB8292, Milli-
pore, Billerica, MA), anti-phosphorylated H2AX (?-H2AX) antibodies
(05-636, Millipore; 2212-1, Epitomics, Burlingame, CA), anti-phosphor-
ylated replication protein A32 (p-RPA32) (3237-1, Epitomics), anti-p53
antibody (GTX102965, GeneTex, Irvine, CA), anti-Flag antibody
(200472, Agilent Technologies, Santa Clara, CA), and anti-?-actin anti-
body (A5441, Sigma-Aldrich, St. Louis, MO).
Lentivirus production and transduction. Lentiviruses were gener-
ated as previously described (36). EPCs were transduced at an MOI of
approximately 10/cell as previously described (9), whereas S1 cells were
transduced at an MOI of 1/cell. S1 cells were preincubated with p53-
shRNA lentivirus by slow rotation at 4°C for 1.5 h before culture at 37°C.
Immunofluorescence analysis. Immunofluorescence (IF) staining
was performed as previously described (39). Texas Red-conjugated don-
key anti-rat antibody (catalog no. 712-076-153) was used for NS1 or 11-
donkey anti-mouse antibody (catalog no. 715-095-151) was used for
?-H2AX (anti-?-H2AX, Millipore) or Alexa Fluor 488-conjugated don-
Rhodamine red-X-conjugated donkey anti-mouse antibody (catalog no.
715-295-150) was used for 7.5-kDa protein or VP1/VP2 detection, and
Alexa Fluor 488-conjugated donkey anti-rabbit antibody (catalog no.
711-546-152) was used for ?-H2AX (anti-?-H2AX, Epitomics) or
munoResearch Laboratories (West Grove, PA).
Images were taken under a confocal microscope (Eclipse C1 Plus,
Nikon) with Nikon EZ-C1 software.
Flow cytometry analysis. Cells were stained and analyzed by flow
cytometry as previously described (11, 36). For cell cycle analysis of the
B19V-Induced DDR Does Not Cause G2/M Arrest
October 2012 Volume 86 Number 19jvi.asm.org 10749
tion (p.i.) or posttransfection (p.tx.) in succession with the rat anti-NS1
antibody, a Cy5-conjugated goat anti-rat antibody (catalog no. 112-176-
143) for detection of NS1, and 4=,6-diamidino-2-phenylindole (DAPI).
For cell cycle analysis of the lentivirus-transduced S1 cells and EPCs, cells
were stained 48 h posttransduction (p.td.) with the mouse anti-Flag anti-
body, followed by a Dylight 649-conjugated goat anti-mouse antibody
(catalog no. 115-496-003) and DAPI.
To determine the level of a DDR, S1 cells were fixed, permeabilized,
rat anti-NS1 antibodies, followed by FITC-conjugated donkey anti-
anti-rat (catalog no. 712-606-153) secondary antibodies for the detection
of ?-H2AX and NS1, respectively. All of the secondary antibodies were
purchased from Jackson ImmunoResearch Laboratories.
All of the samples were analyzed on a three-laser flow cytometer (LSR
II, BD Biosciences) at the Flow Cytometry Core of the University of
Kansas Medical Center. All flow cytometry data were analyzed using
FACSDiva software (BD Biosciences).
Southern blot analysis. Low-molecular-weight (Hirt) DNA was ex-
tracted from treated cells, followed by DpnI digestion and Southern blot-
ting as previously described (26) with M20 DNA as a probe template.
Images were developed and analyzed with a Typhoon 9700 phosphor-
imager (GE Healthcare).
acrylamide gel electrophoresis (SDS-PAGE) was performed as previously
described (13). For Western blotting, cell lysates were prepared and ana-
lyzed as previously described (36).
Individual B19V viral protein does not induce a DDR in EPCs.
We previously reported that B19V infection induces a DDR in
infected EPCs cultured under normoxic conditions (39). Since
hypoxic conditions promote the infection of EPCs by B19V (10),
we cultured EPCs under hypoxic conditions and infected them
with B19V. B19V infection of EPCs cultured under hypoxic con-
ditions also induced a DDR, as evidenced by the phosphorylation
65]), at levels approximately three times higher than those in in-
fected cells cultured under normoxic conditions (Fig. 1A). Nota-
bly, p53 was phosphorylated in B19V-infected EPCs under nor-
moxic conditions and phosphorylated further under hypoxic
Since B19V expresses the nonstructural proteins (NS1, 7.5-
kDa protein, and 11-kDa protein) in addition to the structural
proteins (VP1 and VP2) during infection (37, 64, 78), we exam-
ined whether the expression of the individual viral proteins in-
duced a DDR in EPCs. We expressed the nonstructural proteins
and structural proteins in EPCs via lentiviral transduction. As a
(HU). At 48 h p.td., EPCs were analyzed for the expression of
phosphorylated H2AX and RPA32 by IF staining. B19V-infected
EPCs showed pan-distribution of ?-H2AX (Fig. 1B) and colocal-
ized foci of NS1 with p-RPA32 (Fig. 1C) in the nuclei, which was
consistent with that in infected EPCs cultured under normoxic
the high infectivity of B19V in EPCs cultured under hypoxic con-
ditions (10). In contrast to the formation of nuclear foci during
B19V infection, NS1-transduced EPCs showed neither phos-
phorylation of H2AX nor colocalization of NS1 and p-RPA32
(Fig. 1B and C). A proportion of the NS1 in transduced EPCs
pattern observed during viral infection. The difference in
?-H2AX and p-RPA32 expression between B19V-infected and
NS1-transduced EPCs suggests that B19V NS1 alone is not suf-
ficient to induce the DDR observed during B19V infection.
Similarly, when the 7.5-kDa, 11-kDa, VP1, and VP2 proteins
were expressed in EPCs by their respective lentiviruses, neither
H2AX nor RPA32 was phosphorylated in transduced EPCs
(Fig. 1B and C), whereas HU treatment of control cells induced
the phosphorylation of both H2AX and RPA32.
cells. Hypoxic conditions support the efficient replication of the
B19V dsDNA genome (M20 DNA) in S1 cells at a level approxi-
induces a DDR as well. To this end, S1 cells were transfected with
M20 and a mutant [M20(VP?)] in which the VP2 ORF was
knocked out and neither VP1 nor VP2 was expressed (data not
both wild-type M20-transfected and B19V-infected S1 cells gen-
erated DpnI digestion-resistant bands of ssDNA, monomer RF
(mRF) DNA, and double RF (dRF) DNA on a Southern blot (Fig.
2A). This indicated that B19V M20 DNA replicated in the
transfected cells. The negative control, M20(NS1?)-trans-
bands, confirming that NS1 is essential for B19V DNA replica-
tion. Transfection of mutant M20(VP?) yielded clear mRF and
dRF DNA bands but not ssDNA bands (Fig. 2A, lane 4). Pro-
duction of ssDNA was significantly inhibited in the absence of
capsid proteins during B19V DNA replication. When the pu-
tative endonuclease domain or helicase-A site was mutated, the
M20(endo?) and M20(heli?) mutants did not replicate in
transfected cells, as evidenced by the lack of DpnI digestion-
resistant DNA bands (Fig. 2B, lanes 3 and 4).
The transfection of M20 induced phosphorylation of both
H2AX and RPA32 and induced the formation of NS1 and
p-RPA32 foci in the nuclei of transfected cells, similar to that
observed in B19V-infected S1 cells (Fig. 2D and E). Moreover,
in the nuclei of M20(VP?)-transfected S1 cells. Notably,
M20(endo?)- and M20(heli?)-transfected NS1-positive (NS1?)
cells did not express ?-H2AX (Fig. 2D) or p-RPA32 (Fig. 2E).
with B19V or transfected with M20 and its mutants by flow cy-
expressing cells (Fig. 2F and G), which was significantly higher
than those induced by nonreplicative M20 DNAs [M20(endo?)
Taken together, these results show that replication of B19V M20
B19V DNA replication-induced DDR does not induce G2/M
arrest after transfection. B19V infection of EPCs triggers a DDR
cascade with the activation of all three PI 3-kinases (ATM, ATR,
and DNA-PKcs) (39). It also arrests infected cells at the G2/M
Lou et al.
jvi.asm.orgJournal of Virology
phase (70). ATM- and ATR-mediated signaling is thought to be
the major pathway leading to cell cycle arrest at the G2/M check-
point (35, 61). However, in EPCs the expression of B19V NS1
the B19V DNA replication-induced DDR in G2/M arrest.
We first tried to identify an M20 mutant that replicates but does
not induce significant G2/M arrest in transfected cells. The DNA
ished DNA replication (data not shown). We focused on the C ter-
minus of NS1, particularly the predicted TADs, since the TAD of
minute virus of mice (MVM) NS1 is dispensable for MVM DNA
replication (33). Three TADs were predicted by analyzing the C ter-
minus of NS1 using a TAD prediction program (54) (Fig. 3A). We
ability of the M20-based TAD mutants to trigger a DDR in trans-
fected cells. The results showed that in only the M20(mTAD2)-
transfected (NS1?) cells were both H2AX and RPA32 clearly
phosphorylated but not in cells transfected with M20(mTAD1)
and M20(mTAD3) (Fig. 3C), supporting the notion that B19V
DNA replication is critical for inducing a DDR.
We next tested the ability of wild-type M20 and its mutants to
arrest the cell cycle at G2/M by transfecting them into S1 cells.
FIG 1 B19V viral proteins do not induce a DDR in EPCs cultured under hypoxic conditions. (A) B19V infection induces a stronger DDR in EPCs cultured under
(blue). (C) At 48 h p.i. or p.td., cells were fixed and costained with an antiviral protein antibody (red), an anti-p-RPA32 antibody (green), and DAPI (blue). Confocal
B19V-Induced DDR Does Not Cause G2/M Arrest
October 2012 Volume 86 Number 19jvi.asm.org 10751
FIG 2 Replication of B19V dsDNA genome induces a DDR in S1 cells. S1 cells, which were precultured under hypoxic conditions for 48 h, were infected with
B19V or electroporated with B19V dsDNA genome (M20) or its mutants, as indicated. At 48 h p.i. or p.tx., cells were harvested and analyzed as follows. (A and
B) Southern blot analysis. Hirt DNA was extracted from infected or transfected S1 cells and digested with DpnI followed by Southern blotting. In panel B, M20
DNA (10 ng) was used as the size marker. The smaller band detected coincidentally at the site of the ssDNA is likely degraded M20 DNA. mRF, monomer
replicative form; dRF, double replicative form. (C) Diagram of the mutations in the endonuclease motif and helicase-A site of the NS1 protein. Amino acid
sequences in the endonuclease and helicase-A domains, and mutations are shown in red. (D and E) IF staining of the DDR markers. Cells were costained with
drawn based on the secondary antibody-only control (mock) for a cutoff between the NS1-negative (NS1?) and -positive (NS1?) populations. Both the
NS1-positive rate (%) and the mean fluorescence intensity (MFI) of NS1 expression in the NS1?population are presented in each plot. For ?-H2AX staining,
levels of ?-H2AX are shown as the MFI both in the NS1?and NS1?cell populations of each group in the middle and right-hand histograms, respectively. The
the NS1?to that for the NS1?population and is represented as the mean plus or minus the standard deviation from at least three independent experiments. P
values were determined using Student’s t test.
jvi.asm.orgJournal of Virology
within the C terminus of B19V NS1. The TADs within B19V NS1 were predicted by analyzing the NS1 amino acid sequence using the 9aaTAD program (54).
Three predicted TADs are shown with the mutated amino acids indicated in red above the TADs. (B) Southern blot analysis of transfected DNA replication. S1
cells cultured under hypoxic conditions were transfected with M20, M20(mTAD1), M20(mTAD2), or M20(mTAD3). At 48 h p.tx., cells were collected for Hirt
DNA preparation followed by Southern blotting. (C) IF staining of the DDR markers. At 48 h p.tx., the transfected cells were costained with anti-NS1 (red),
cell cycle. M20 and its indicated mutants were electroporated into S1 cells. B19V-infected S1 cells served as a positive control for NS1 detection and cell cycle
analysis by flow cytometry. The y axes for all the plots are in arithmetic scale. The x axes for the NS1-staining plots are in logarithmic scale, while those for the
(%) of the cells in each phase of the cell cycle is shown. (E) Statistical analysis. Data for the percentage of cells at the G2/M phase (G2/M%) were obtained from
at least three independent experiments and are presented as the mean plus or minus the standard deviation. The P value was determined using Student’s t test.
B19V-Induced DDR Does Not Cause G2/M Arrest
October 2012 Volume 86 Number 19jvi.asm.org 10753
Notably, the M20 mutants that did not replicate induced clear
G2/M arrest at levels similar to or higher than those observed in
the wild type, with the percentage of cells in G2/M phase ranging
from 63.1% to 88.6% in NS1?cells (Fig. 3D and E). More impor-
tantly, the replicative TAD mutant, M20(mTAD2), significantly
decreased the number of cells at the G2/M phase compared with
that of the wild-type M20 control (32.8% vs. 57.7%, respectively)
Collectively, these results show that replication of the B19V
dsDNA genome is not required to induce G2/M arrest in trans-
fected S1 cells. Since replication of the B19V dsDNA genome is
essential for inducing a DDR, we conclude that the DDR is dis-
pensable for G2/M arrest of the cells in which B19V DNA repli-
cates. The results also suggest that B19V NS1 alone is sufficient to
arrest the cell cycle at G2/M during infection.
S1 cells and EPCs. To examine the sole role of NS1 in inducing
G2/M arrest, we expressed only wild-type NS1 and the NS1 mu-
duction in S1 cells and EPCs. Transduction of Lenti-½ITR-P6-
NS1, in which expression of wild-type NS1 is driven by the native
B19V P6 promoter (55), recapitulated G2/M arrest to the level
the transduction of lentiviruses expressing the NS1 mutants ex-
similar to that induced by the expression of the RFP control
in S1 cells, both B19V infection and transduction of lentiviruses
expressing wild-type NS1 and the NS1 mutants, NS1(endo?),
NS1(heli?), and NS1(mTAD3), induced over 90% of NS1?cells
at the G2/M phase (Fig. 4B). Notably, the expression of
G2/M, a level close to that induced by expression of the RFP con-
trol (35.0%) (Fig. 4D). We noticed that the expression of RFP in
the context of Lenti-½ITR-P6-RFP induced a higher number of
cells at G2/M (approximately 40%) than G2/M-phased cells (ap-
sequence (5=-GTTTTGT-3=) exits in the B19V P6 promoter and
inhibits proliferation of EPCs by arresting the cell cycle at the S
and G2/M phases (28). We also observed that a lentiviral vector
bearing a B19V sequence of nucleotides 187 to 615 (contains
½ITR and the core P6 promoter) arrested the cell cycle at G2/M
(data not shown).
Taken together, these results confirm that the expression of
B19V NS1 alone is sufficient to induce nearly complete G2/M ar-
cells and EPCs.
The p53-mediated pathway is not required for G2/M arrest
induced by B19V infection or transfection of the B19V dsDNA
p53 at Ser15(3, 60). The cyclin-dependent kinase Cdk1, which
controls the G2/M transition, is inhibited by the product of the
p53-targeting gene, p21 (6). Since B19V NS1 transactivates the
expression of p21 (45) and p53 is phosphorylated at Ser15during
B19V infection of EPCs (70) (Fig. 1A), we decided to examine the
role of p53 in B19V-induced G2/M arrest.
shRNAs, which proved to be very difficult (data not shown). For-
tunately, nearly perfect p53 knockout was obtained in S1 cells
found that the percentage of p53-deficient NS1?cells (either in-
fected with B19V or transfected with M20) at the G2/M phase did
not used to induce G2/M arrest during infection or transfection.
arrest at G2/M than B19V infection (56.8% vs. 90.6%) (Fig. 5B);
however, knockout of p53 did not ameliorate G2/M arrest signif-
icantly under either condition (Fig. 5C).
In this study, we report that the replication of the B19V dsDNA
identified an M20 mutant, M20(mTAD2), containing a mutated
transactivation domain within NS1, which replicated well in S1
cells but did not arrest the cell cycle at the G2/M phase. In agree-
ment with this result, the NS1 harboring the TAD2 mutation did
not induce significant G2/M arrest in either EPCs or S1 cells by
lentiviral transduction. Finally, we showed that the p53-mediated
pathway is not involved in G2/M arrest during B19V infection.
Thus, our results provide evidence that the DDR induced during
B19V infection does not contribute to G2/M arrest and confirm
that NS1 alone is sufficient to induce the G2/M arrest in the con-
text of the B19V P6 promoter.
A DDR is a response by the cellular surveillance network that
senses and repairs damaged DNA and is required to maintain
genome integrity (14). During viral infection, a DDR plays a role
in the intrinsic antiviral mechanism to eliminate the invasion of
the viral genome; however, in some cases, the virus exploits the
DDR signaling mechanism to promote its own replication (71).
B19V infection of both EPCs and S1 cells typically arrests the cell
cycle at G2/M (39, 47, 70). Preventing replication of the B19V
dsDNA genome by mutating the endonuclease domain or heli-
case-A motif within NS1, which prohibits the DDR, did not re-
duce the percentage of cells arrested at G2/M; however, mutating
TAD2 could result in a replication-competent M20 mutant but
with reduced G2/M arrest. NS1 alone is sufficient to cause G2/M
the mechanism of a DDR only to promote viral DNA replication
(39), and the NS1-induced G2/M arrest is dispensable for viral
B19V infection warrants further investigation. The fact that the
p53 pathway is not used by B19V NS1 to arrest the cell cycle indi-
cates that the TAD2 of NS1 does not transactivate p53-regulating
genes. NS1 expression in EPCs upregulates 44 genes and down-
regulates 28 genes that are involved in cell cycle regulation (70).
Therefore, we hypothesize that TAD2 of NS1 may play an impor-
tant role in regulating one or several of these genes. The NLS
domain of NS1 is thought to be critical for inducing G2/M arrest,
(70); however, mutation of the NLS, which prevents NS1 trans-
port into the nucleus (70), may also prevent the function of the
Lou et al.
jvi.asm.org Journal of Virology
G2/M arrest induced by NS1 during B19V infection, which is in-
dependent of the DDR and p53 activation.
during infection (8, 34, 71), particularly in the large T antigen of
polyomaviruses (21, 49, 77) and the Vpr of HIV (76). In parvovi-
ruses, the replication of the AAV2 genome during coinfection
DDR (17, 59). We reported that DNA replication of the minute
FIG 4 NS1 alone is sufficient to induce G2/M arrest in S1 cells and EPCs. (A and B) Flow cytometry analyses of S1 cells and EPCs transduced with lentiviruses.
serves as a lentiviral control. At 48 h p.i. or p.td., cells were costained with anti-NS1 and Cy5-conjugated secondary antibodies (for B19V-infected cells) or
analyses were performed as described in Fig. 3D. (C and D) Statistical analysis. Statistical data of the G2/M% were obtained from at least three independent
experiments in S1 cells (C) and EPCs (D), indicated as the mean plus or minus the standard deviation. The P values were determined using Student’s t test.
B19V-Induced DDR Does Not Cause G2/M Arrest
October 2012 Volume 86 Number 19 jvi.asm.org 10755
virus of canines (MVC) genome is responsible for an MVC-in-
duced DDR rather than the viral proteins (38). The MVM also
induces a DDR during infection (1, 56), but the small nonstruc-
tural protein NS2 is dispensable (56). The AAV2 Rep78 induces a
DDR by introducing nicks in the cellular chromatin (4), but it is
only a minor contributor to the DDR induced during AAV2 in-
fection (59). The present study shows that B19V NS1, the small
nonstructural proteins (7.5-kDa and 11-kDa proteins), and the
capsid proteins failed to induce an obvious DDR in EPCs when
the possibility of the combined expression of B19V proteins,
which is the case during B19V infection, and transfection of the
replication-competent M20 DNAs inducing a DDR. Knockout of
replication in S1 cells (data not shown). We speculate that a com-
bined expression of B19V proteins supports B19V DNA replica-
tion to the highest extent, which induces a maximum DDR. This
actually is true for the NS1 protein. NS1 is essential for B19V
DNA replication (Fig. 2A), but NS1 alone could not induce a
DDR (Fig. 1B and C). Taken together, our results support the
conclusion that the DDR induced during parvovirus infection
is mediated mainly through replication of the viral genome, for
which NS1 is essential.
Because EPCs are difficult to transfect (13), we delivered the
of the B19V dsDNA genome, even in the absence of ssDNA pro-
indicate that replication of the B19V dsDNA genome during in-
fection of EPCs initiates the DDR independently of the produc-
B19V and other parvoviruses have hairpin termini at both ends
(20). The gap between the termini displays a structure of a single-
strand break (SSB) that is recognized by ATR (15). Since it was
FIG 5 The p53 pathway is not necessary for B19V-induced G2/M arrest. S1 cells cultured under normoxic conditions were transduced with p53-shRNA- and
scramble-shRNA (scr-shRNA)-expressing lentiviruses, which also expressed GFP (9), and then cultured under hypoxic conditions for 48 h. The cells then were
not transduced with lentivirus. The arrow indicates the p53 band. (B) Flow cytometry analysis of the cell cycle. At 48 h p.i. or p.tx., cells were costained with the
shRNA-treated groups, shRNA-expressing cells were selected based on the GFP signal expressed by the lentiviral vector and shown in the left-hand histograms.
(C) Statistical analysis of the cell cycle results. The ratio of the percentage of cells at G2/M (G2/M%) in the NS1?population to that in the NS1?population in
indicated as the mean plus or minus the standard deviation. The P values were determined using Student’s t test.
Lou et al.
jvi.asm.org Journal of Virology
difficult to detect the ssDNA viral genome in the nucleus upon
B19V infection (data not shown), we have not provided direct
evidence that the B19V ssDNA genome elicits a DDR. However,
when UV-inactivated B19V was used to infect EPCs at an MOI of
neither ?-H2AX nor p-RPA32 was detected in infected cells (data
not shown). It has been reported that infection of UV-inactivated
AAV at a high MOI (5,000 gc/cell) elicited a DDR by mimicking
the stalled replication fork (29). Further studies are warranted to
determine the nature of the B19V ssDNA genome as an SSB sub-
strate for ATR recognition.
B19V induces a DDR via replication of the RF genome, which
involves replication intermediates (27) rather than via NS1 ex-
pression alone. The replication of parvovirus DNA follows the
displacement step (19). Therefore, the formation of a replication
origin complex at the replication origin, or other steps, such as
nicking at the terminal resolution site by NS1 followed by un-
winding of the RF DNA or hairpin by the helicase activity of NS1
(which resembles a stalled replication fork), is likely the key for
triggering a DDR during virus infection. It is possible that ATR
recognizes the nicked site within the terminal sequence of the
the displaced ssDNA, which mimics a stalled replication fork.
Thus, our observations have laid a solid foundation for further
study of how replication of the ssDNA genome of autonomous
parvovirus induces a DDR during virus infection.
S or G2/M phase checkpoint, is a classic strategy for infecting vi-
ruses to exploit cellular components/mechanisms to aid viral ge-
nome replication (12, 23). MVM and MVC infections activate
ATM-mediated signaling, which arrests infected cells at the S and
G2/M phases; the DDR then directly leads to cell cycle arrest (1,
infection, which results in G2/M arrest through ATR-checkpoint
kinase 1 (Chk1) signaling (66). It is intriguing that B19V DNA
replication-induced DDR does not result in G2/M arrest, even
fore, our observations suggest multiple roles for virus infection-
triggered DDR. In some cases, the DDR induces cell cycle arrest,
which in turn facilitates virus replication or kills infected cells. In
other cases, the DDR is directly involved in promoting DNA rep-
lication without arresting the cell cycle.
In summary, we have demonstrated that replication of the
dispensable for triggering cell cycle arrest during B19V DNA rep-
lication. However, the NS1 protein was instrumental in G2/M ar-
rest, which did not require the putative activity of endonuclease
and helicase but did require TAD2 function. Furthermore, this
process was independent of the p53 pathway. Further studies are
needed to examine how TAD2 functions to induce G2/M arrest
egy employing NS1 to overcome difficulties in arresting the cell
cycle in rapidly proliferating EPCs. The reason why the induced
ATR and ATM signaling, did not significantly result in G2/M ar-
rest will be an interesting topic for future study.
This work was supported in full by PHS grant R01 AI070723 from the
NIAID and grant R21 HL106299 from the NHLBI to J.Q.
We are indebted to Susan Wong at the Hematology Branch, NHLBI,
NIH, for helping culture CD34?human hematopoietic stem cells.
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